Genomes & Genomics. Genome, the entire genetic complement of an organism Genomics, research that addresses all or a substantial portion of an organisms. - презентация

1 Genomes & Genomics

2 Genome, the entire genetic complement of an organism Genomics, research that addresses all or a substantial portion of an organisms genome Includes physical mapping & sequencing of all or a large part of a genome or chromosome

3 Why Study Genomes of Different Organisms? To understand the genetics behind diseases (Homo sapien & Canis familiarus) To learn more about human pathogens & how to prevent or treat their infections (Clostridium tetani, Bacillus anthacis, & Haemophilus influenzae) Understand & improve the genetics of commercial organisms (Lactococcus lactis, Oryza sativa, Bos taurus, & Gallus gallus) To discover the workings of unusual or odd organisms (Bdellovibrio bacteriovorus & Deinococcus radiodurans) To understand phyolegeny

4 How Many Genomes Have Been Sequenced? CompletedDraftIn Progress Eukaryote Archaea Eubacteria Viral 1703 (NCBI 9/4/07)

5 How Do We Measure a Genome? 1 base=1 nucleotide=1basepair (bp) 1000bases=1kilobase (Kb) 1000kb=1megabase (Mb) 1000mb=1gigabase (Gb)

6 Genome Sizes (haploid) OrganismGenome in Mb E. coli4.64 Yeast12 Nematode97 Fruit Fly170 Pufferfish345 Human3200 Lungfish129000

7 basepairs Amount of DNA in a Genome Does Not Correlate with Complexity

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9 Genomes Are Organized Into Chromosomes Human Fruit Fly

10 Chromosome Number Is Species Specific Diploid Number 2n Human46 Mouse40 Fruit Fly8 Dog78 Arabidopsis10 Corn20 Yeast32 Crayfish200

11 How many genes do we have? Original estimate was between to genes We now think human have ~ genes How does this compare to other organisms? Mice have ~ genes Pufferfish have ~ gene The nematode (C. elegans), has ~ Yeast (S. cerevisiae) there are ~6000 genes The microbe responsible for tuberculosis has ~4000

12 Gene Spacing in Various Species

13 Even the Amount of DNA a Gene Spans Differs Amongst Species

14 Comparative Genomics

15 Yeast 70 human genes are known to repair mutations in yeast Nearly all we know about cell cycle and cancer comes from studies of yeast Advantages: fewer genes (6000) few introns 31% of yeast genes give same products as human homologues

16 Drosophila nearly all we know of how mutations affect gene function come from Drosophila studies We share 50% of their genes 61% of genes mutated in 289 human diseases are found in fruit flies 68% of genes associated with cancers are found in fruit flies Knockout mutants Homeobox genes

17 C. elegans 959 cells in the nervous system 131 of those programmed for apoptosis apoptosis involved in several human genetic neurological disorders Alzheimers Huntingtons Parkinsons

18 Mouse known as mini humans Very similar physiological systems Share 90% of their genes

19 What is the rest of the human genome made up of? Regulatory regions of DNA that turn genes on or off Repetitive DNA sequences: Tandem Repetitive Sequences (~10%) Microsatellite DNA: 2 to 4bp long repeats Minisatellite DNA: 20bp or longer repeats Macrosatellite DNA: megabase long repeats Transposable elements SINEs and LINEs 35% Retroviral fossils

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21 Genetic vs. Physical Mapping

22 Genetic mapping based on genetic techniques, maps show the positions of diseases or traits based on recombination frequencies Genetic techniques include cross-breeding experiments or, the examination of family histories (pedigrees) Physical mapping uses molecular biology techniques to examine DNA molecules directly to construct maps showing the positions of sequence features, including genes Physical techniques include DNA restriction enzyme analysis & fluorescent tagging of chromosomal regions

23 Genetic Map showing the location of disease genes on human chromosome 4

24 Human chromosomes stained to show bands of different DNA These bands are the roughest markers for physical mapping

25 Fluorescent Labeling of Chromosomes

26 Types of Physical Maps For Chromosome 21

27 The more markers better the resolution, the more useful the map

28 DNA Sequencing

29 Polyacrylamide gel electrophoresis can resolve ssDNA molecules that differ in length by just one nucleotide A banding pattern is produced after separation of ssDNA molecules by denaturing polyacrylamide gel electrophoresis

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31 Automatic Sequencing Machines use fluorescent dyes

32 Fluorescent Dye Dideoxy-sequencing

33 DNA Sequencers in Action

34 First Complete Sequence of a Free-Living Organism

35 1995, the Haemophilus influenzae genome sequenced Genome size=1830 kb 1 st genome sequenced using the shotgun method 28,643 sequencing experiments totaling 11,631,485 bp This equaled 6x the length of the H. influenzae genome Sequence assembly 30 hrs on a computer with 512 Mb of RAM Resulted in 140 lengthy contiguous sequences Each sequence contig represented, non-overlapping portion of the genome

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39 1 st proposed by the DoE 1984 By 1990, the Human Genome Project was launched The Human Genome Organization (HUGO) was founded to provide a forum for international coordination of genomic research The program was proposed to include: The creation of genetic & physical maps to be used in the generation of a complete genome sequence Human Genome Project

40 First Steps of the Human Genome Project 1) Construct genetic & physical maps of the haploid human & mouse genomes These would provide key tools for identification of disease genes and anchoring points for genomic sequence 2) Sequence the yeast and worm genomes, as well as targeted regions of mammalian genomes

41 Sequencing Plan of HUGO 1) Isolate each human chromosome 2) Physical mapping of each chromosome The banding pattern of visible through staining Location of known genes already mapped Location of restriction enzyme sites Chromosome fragmented into large pieces of DNA and inserted into BAC or YAC libraries Fragments overlap such that they can be ordered into a rough assembly of the chromosome DNA from 5 humans 2 males, 3 females 2 caucasians, one each of asian, african, hispanic

42 Each YAC or BAC is fragmented into smaller 1 to 2 kb pieces of DNA which are sequenced Each of these fragments slightly overlaps with each other A computer takes the DNA sequences & looks for regions of overlap these are connected to form a sequence contig for the entire BAC or YAC The sequence of all the YACs or BACs are assembled through the same process to give the sequence of the chromosome This is repeated for all 22 chromosomes plus the X & Y

43 Hierarchical Shotgun Approach Separate Individual Chromosomes

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45 Chromosome 11 BACs

46 1999, Celera Genomics, set out to sequence the human genome using a whole-genome shotgun method - more riskier - goal to patent some seq. There would be no isolation of individual chromosomes & no subcloning into BACs or YACs They skipped straight to the 1 to 2 kb fragments The $300 million Celera effort was intended to proceed at a faster pace and at a fraction of the cost of the roughly $3 billion HUGO project. Dr. Craig Venter (founder) Celera Genomics Human Genome Whole- Genome Shotgun Method

47 14.8-billion bp of DNA sequence was generated over 9 months This equaled 5x the human genome Resulting sequence contigs spanned >99% of the genome In March 2000, President Clinton announced that the genome sequences could not be patented, and should be made freely available to all researchers. The statement sent Celera's stock plummeting. The competition proved to be very good for the project, spurring the public groups to modify their strategy in order to accelerate progress. In February 2001 Celera Genomics published their draft of the human genome in the journal Science The same month HUGO published its draft of the human genome in the journal Nature The rivals initially agreed to pool their data, but the agreement fell apart when Celera refused to deposit its data in the unrestricted public database GeneBank.

48 Hierarchical Shotgun Approach Whole-Genome Shotgun Approach

49 Celera took multiple copies of the genome fragmented them into 1 to 2kb fragments which where sequenced without concern for what chromosome they belonged to

50 What did they learn? 1.1% of the genome is spanned by exons 24% is in introns 75% of the genome is intergenic DNA A random pair of human haploid genomes differs on average at a rate of 1 bp per 1250 bp

51 Preliminary Functional Analysis of > genes > (41%) have no known function S. Barnum, 2005 Biotechnology, An Introduction. Brookes/Cole

52 Diploid Genome Sequence of an Individual Human On September 4th, 2007, a team led by Craig Venter, published his (ovn) complete DNA sequence, unveiling the six-billion-letter genome of a single individual for the first time. 44% of known genes had one or more alterations >0.5% variation between two haploid genomes

53 How Do We Differ? Total of 4.1 million DNA variations 3.2 million single nucleotide changes 53,800 block substitutions (2 to 206bp) 292,000 heterozygous insertion/deletions (1 to 571bp) 559,000 homozygous insertion/deletions (1 to 82,711bp) 90 inversions Numerous duplications & copy number variations

54 The UCSC Genome Browser

55 The browser takes you from early maps of the genome...

56 ... to a multi-resolution view...

57 ... at the gene cluster level...

58 ... the single gene level...

59 ... the single exon level...

60 ... and at the single base level caggcggactcagtggatctggccagctgtgacttgacaag caggcggactcagtggatctagccagctgtgacttgacaag

61 Other –omics Proteomics Transcriptomics Metabolomics Glycomics Epigenomics Metagenomics